The aim of this project is to determine the influence of specific structural features of the 5'-terminal portion of eukaryotic mRNA on initiation of protein synthesis in vitro. We have extended our studies using tobacco mosaic virus RNA and rabbit globin mRNA. These RNAs were enzymatically 'decapped' by nucleotide pyrophosphatase from potato which resulted in the removal of greater than 95% of 5'-terminal m7G. Translation of control and 'decapped' mRNAs in a wheat germ or reticulocyte lysate showed dependence on 5'-terminal m7G, especially at high potassium ion concentrations. We have analyzed the 5'-terminal m7G, cap structures of sub-genomic RNA fractions from turnip yellow mosaic virus (TYMV) RNA to determine whether there is 5'heterogeneity which would be indicative of minor species of mRNA coding for non-structural proteins. Enzymatic analysis of TYmV RNA suggests that in addition to m7GpppGmpA, and m7GpxpAmpA, the expected structures for the genome RNA and coat protein mRNA, there is one other compound, whose structure is currently under investigation.